scholarly journals Myc and the Warburg Effect

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-12-SCI-12
Author(s):  
Stefano Casola ◽  
Gabriele Varano ◽  
Laura Perucho ◽  
Marianna Ossorio ◽  
Federica Zanardi ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Metabolic Pathways ◽  
Gene Networks ◽  
Conflicts Of Interest ◽  
Lymphoproliferative Disorders ◽  
B Cell Malignancies ◽  
Lymphoma Cells ◽  
Bcr Signaling

Abstract Mature B cells recognize and respond in a highly-specific fashion to a multitude of environmental antigens through membrane-bound immunoglobulins forming together with the Igα and Igβ proteins a functional unit called the B cell antigen receptor (BCR). Through a complex network of effector molecules, the BCR transforms environmental signals into biochemical reactions which are responsible for highly codified cellular responses affecting survival, proliferation, migration and terminal differentiation of B cells. Surface BCR expression is conserved in most types of B cell malignancies arising from mature B cells. This observation, together with genetic and biochemical evidence pointing to sustained BCR signaling in different types of B cell neoplasms represents the rationale for the current use of pharmacological inhibitors of BCR signaling to treat several forms of B lymphoproliferative disorders. Nevertheless, our understanding of how the BCR influences malignant B cell behavior remains poorly understood. In an attempt to fill this knowledge gap, we engineered a mouse model to monitor the effects of acute ablation of the BCR in highly-aggressive MYC-driven lymphomas. Inducible BCR ablation did not, per se, prevent the outgrowth of receptor-less MYC lymphoma cells both in vitro and in vivo. Instead, BCR loss weakened the fitness of the malignant B cells leading to the rapid elimination of BCR-less tumor cells in the presence of their BCR-expressing counterparts (Varano et al., 2017). Through the integration of data generated from genomics, metabolomics and bulk/single cell transcriptomics analyses, comparing BCR-deficient lymphoma cells to their proficient counterparts, we have started to elucidate the gene networks and metabolic pathways influenced by BCR expression that sustain competitive fitness of MYC-transformed lymphoma B cells. Data from CRISPR/Cas9-mediated disruption of candidate fitness genes in primary malignant B cells will be presented. In support of the findings in the mouse model, we will provide evidence that BCR-less malignant B cells are spontaneously generated during tumor progression in several forms of human B cell lymphoproliferative disorders, establishing a possible Achilles heel of anti-BCR therapies. Finally, we will report possible strategies enabling the clearance of BCR-less lymphoma cells, taking advantage of their acquired addiction to specific signaling and metabolic pathways. Our results shed light on the coordinated regulation of signaling and metabolism imposed on malignant B cells by BCR expression/signaling and provide indications for improved treatment options to fight several forms of mature B cell malignancies. Reference: Varano G, Raffel S, Sormani M, Zanardi F, Lonardi S, Zasada C, Perucho L, Petrocelli V, Haake A, Lee AK, Bugatti M, Paul U, Van Anken E, Pasqualucci L, Rabadan R, Siebert R, Kempa S, Ponzoni M, Facchetti F, Rajewsky K, Casola S. Nature. 2017; 546:302-306. Disclosures No relevant conflicts of interest to declare.

TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3665-3665
Author(s):  
Feng Guo ◽  
Peng Zhou ◽  
Liang Ma
Keyword(s):  
B Cells ◽  
B Cell ◽  
Expression Vector ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Activity Assay ◽  
Lymphoma Cells ◽  
Aberrant Expression ◽  
Wide Range ◽  
P100 Processing

Abstract Abstract 3665 Poster Board III-601 Introduction Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear. Methods Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector. Results In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells. Conclusions Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells. Disclosures: No relevant conflicts of interest to declare.

Overexpression of the Protein Tyrosine Phosphatase Lyp Reduces the Responsiveness of Chronic Lymphocytic Leukemia B-Cells to B-Cell Receptor Ligation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 800-800
Author(s):  
Roberto Negro ◽  
Pablo G Longo ◽  
Michela Tarnani ◽  
Stefania Gobessi ◽  
Luca Laurenti ◽  
...  
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Signaling Molecules ◽  
Negative Regulator ◽  
Nucleotide Sequence Analysis ◽  
Aberrant Expression ◽  
Bcr Signaling

Abstract Abstract 800 CLL B cells display many features that suggest a role for antigen stimulation in the development and progression of the disease. These include the expression of stereotyped B-cell receptors (BCRs), the association between IgVH gene mutation status and prognosis, and the gene-expression profile of antigen-stimulated B cells. In addition, CLL B cells have other BCR-related features that distinguish them from normal B lymphocytes, such as lower levels of surface Ig, less efficient BCR signal transduction and increased basal activity of the proximal BCR signaling molecules Lyn and Syk. We have now investigated whether any of these features are related to aberrant expression or function of the phosphatases SHP-1, SHP-2 and Lyp (PTPN22), which regulate the amplitude and duration of the BCR signal by dephosphorylating various components of the BCR signal transduction unit. These phosphatases are also interesting because mutated or polymorphic variants have been linked to various malignant or autoimmune diseases. We started our study by performing nucleotide sequence analysis of the complete coding region of SHP1, SHP2 and Lyp in 8, 21 and 29 CLL B cell samples, respectively. Overall, only two mutations were identified (an R527C substitution in SHP2 and a Q456E substitution in Lyp, each in a single patient), suggesting that these phosphatases are infrequently mutated in CLL. The previously reported Lyp polymorphisms R620W and R263Q were observed in 2 additional cases. We next investigated expression of these phosphatases in purified CLL and normal B cells by immunoblotting. Expression of SHP1 and SHP2 was relatively uniform in the different CLL B-cells samples (n=42) and was not different from normal B cells (n=4). In contrast, expression of Lyp was markedly higher in most CLL samples, with 35 of the 49 investigated cases exhibiting 2 to more than 10 fold higher levels than normal B cells (n=5) (CLL, mean Lyp levels 4.7, SD +/−3.7; normal B cells, mean Lyp levels 0.9, SD +/−0.1, P=0.022). The mean Lyp levels were somewhat higher in U-CLL than M-CLL (6.0 vs. 3.9) and ZAP-70-positive than ZAP-70-negative cases (5.6 vs. 4.7), but these differences were not statistically significant. Analysis of Lyp expression in various lymphoma B-cell lines (n=9) also did not reveal significant differences with respect to normal B-cells, suggesting that Lyp overexpression is a specific feature of CLL. To determine what are the consequences of Lyp overexpression on BCR signaling, we downregulated Lyp in primary CLL B-cells by RNA interference and investigated activation of BCR signaling molecules following sIgM crosslinking. Downregulation of Lyp resulted in a substantial increase in BCR-induced phosphorylation of Lyn (Y397), Syk (Y352), BLNK (Y84) and ERK (T202/Y204), suggesting that overexpression of this phosphatase may be at least partially responsible for the lower BCR signaling capacity of CLL B-cells. Since Lyp expression can be induced in resting T cells by activation with anti-CD3, we investigated whether BCR stimulation will have a similar effect on CLL B-cells. A two-fold increase in Lyp levels was observed after 24 hours of sustained BCR stimulation with immobilized anti-IgM, whereas transient stimulation with soluble anti-IgM resulted in a 20% decrease in Lyp levels. These effects were specific for Lyp, since no such changes were observed in the expression of SHP1 and SHP2. In summary, this study shows that CLL B-cells specifically overexpress the phosphatase Lyp, and important negative regulator of BCR signaling that has been implicated in the pathogenesis of several common autoimmune diseases. Given the observation that Lyp can be induced by sustained BCR engagement and in view of recent findings that Lyp is also overexpressed in anergic B cells, these data further support the notion that CLL cells are continuously exposed to (auto)antigen in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sodium stibogluconate and CD47-SIRPα blockade overcome resistance of anti-CD20-opsonized B cells to neutrophil killing

2021 ◽  
Author(s):  
Dieke J. van Rees ◽  
Maximilian Brinkhaus ◽  
Bart Klein ◽  
Paul Verkuijlen ◽  
Anton T.J. Tool ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Target Cells ◽  
Sodium Stibogluconate ◽  
B Cell Malignancies ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
B Lymphoma Cells ◽  
Anti Cd20

Anti-CD20 antibodies, like rituximab, are broadly used to treat B cell malignancies. These antibodies can induce various effector functions, including immune cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Neutrophils can induce ADCC towards solid cancer cells by trogoptosis, a cytotoxic mechanism known to be dependent on trogocytosis. However, neutrophils appear incapable of killing rituximab-opsonized B lymphoma cells. Nevertheless, neutrophils do trogocytose rituximab-opsonized B lymphoma cells, yet this only reduces CD20 surface expression, and is thought to render tumor cells therapeutically resistant to further rituximab-dependent destruction. Here, we demonstrate that resistance of B lymphoma cells towards neutrophil killing can be overcome by a combination of CD47-SIRPα checkpoint blockade and sodium stibogluconate (SSG), an anti-leishmanial drug and documented inhibitor of the tyrosine phosphatase SHP-1. SSG enhanced neutrophil-mediated ADCC of solid tumor cells, but enabled B lymphoma cell trogoptotic killing, by turning trogocytosis from a resistance-contributing mechanism into a cytotoxic anti-cancer one. The killing in the presence of SSG required both antibody opsonization of the target cells, as well as disruption of CD47-SIRPα interactions. These results provide a more detailed understanding of the role of neutrophil trogocytosis in antibody-mediated destruction of B cells and clues on how to further optimize antibody therapy of B cell malignancies.

scholarly journals Cooperation between SYK and ZAP70 Kinases As a Driver of Oncogenic BCR-Signaling in B-Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3922-3922
Author(s):  
Teresa Sadras ◽  
Jevon Cutler ◽  
Julia Aguade-Gorgorio ◽  
Zhengshan Chen ◽  
Kadriye Nehir Cosgun ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Kinase Domain ◽  
Leukemic Cells ◽  
B Cell Malignancies ◽  
Linker Region ◽  
Sh2 Domains ◽  
Bcr Signaling ◽  
Survival Signals

Abstract The spleen tyrosine kinase (SYK) and ζ-associated protein of 70 kD (ZAP70) tyrosine kinases play critical roles in proximal signal transduction of B-cell (BCR) and T-cell receptors (TCR), respectively. The highly similar SYK and ZAP70 kinases share a common structure composed of two tandem SH2 domains and a carboxy-terminal kinase domain. A linker region, termed interdomain B, connects the SH2 domains to the kinase domain and is important for kinase activation. Despite their conserved structure, SYK and ZAP70 are expressed in a largely mutually exclusive manner and play analogous roles in BCR- and TCR-signaling. Cross-lineage activation of ZAP70 in B cells was previously identified in chronic lymphocytic leukemia (CLL), which is characterized by clonal accumulation of malignant CD5+ B-cell cells that retain dependency on the BCR for survival signals. Nearly half of CLL cases show co-expression of SYK and ZAP70, and these patients have an aggressive disease course and a poor prognosis. Our analysis shows that in addition to CLL, aberrant ZAP70 expression occurs in other B-cell malignancies, e.g. TCF3-PBX1 pre-B ALL and B-cell lymphoma subsets that depend on survival signals from a functional (pre-) BCR. These findings suggest that interactions between SYK and ZAP70 may function to fine-tune strength of oncogenic BCR-signaling. To test this hypothesis, we have used a combination of molecular and proteomic approaches. We studied mechanisms by which ZAP70 integrates into BCR-mediated signals, and how the function of ZAP70 in B-cells differs from its native role downstream of the TCR. We demonstrate that ectopically expressed SYK and ZAP70 proteins are constitutively phosphorylated in BCR-ABL1+ B-ALL cells, but these induce distinctive signaling thresholds. CRISPR-mediated deletion of SYK or ZAP70 in leukemic cells further revealed that SYK and ZAP70 regulate unique signaling pathways in B-cells. We also demonstrate that ZAP70 is activated following BCR stimulation of lymphoma cells, and SYK/ZAP70 co-expressing cells display enhanced BCR signaling. Interestingly, enhanced BCR signaling was also observed in cells engineered to express an alternative splice variant of SYK (SYK-S). This shorter isoform of SYK, lacks a 23 amino-acid insert in the interdomain-B linker region, which is also absent in ZAP70, and may define unique protein-interactions that modulate signaling outcome. To elucidate the differential interactome of SYK, SYK-S, and ZAP70 we performed proximity-dependent biotin identification (BioID) experiments in B-cells following BCR-activation to capture the core signalling networks of these kinases in leukemic cells. In addition to expected BCR components including BLNK, PTPN6 and CBL we identified novel SYK and ZAP70 associated molecules including IKZF3, LAT2 and WAS which may play important roles in the survival of BCR-dependent malignancies. Importantly our findings highlight a role for ZAP70 in oncogenic BCR-signaling and suggest that ZAP70 promotes oncogenic BCR-signaling by limiting the ability of the BCR to induce negative B-cell selection and cell death. Disclosures No relevant conflicts of interest to declare.

scholarly journals ZAP-70 Expression in Non Tumoral B Cells: Role in B Tolerance Breakdown?

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1114-1114
Author(s):  
Mickaël Martin ◽  
Anne Marie Knapp ◽  
Dana Ghergus ◽  
Fabien Delmotte ◽  
Laurent Vallat ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Rt Pcr ◽  
Variable Regions ◽  
Conditional Expression ◽  
Bcr Signaling ◽  
Tolerance Breakdown ◽  
First Time

Abstract Abnormal expression of the tyrosine kinase ZAP-70 by tumoral B cells in chronic lymphocytic leukemia (CLL) is associated with bad prognosis, related to B cell receptor (BCR) hypersignalling, clonal expansion and autoimmune cytopenia (AIC) occurrence, these latter being mostly induced by polyclonal IgG from the residual non tumoral B cells. We previously shown that ZAP-70 is expressed by these non tumoral B cells in CLL, positively associated with its expression in CLL B cells and with AIC occurrence (Ghergus et al. Poster ASH 2017). Here, we show for the first time a potential role of ZAP-70 expression in tolerance breakdown in CLL and in an original knock in mouse model overexpressing ZAP-70 conditionally in B cells. First, to assess a potential molecular link between ZAP-70+ CLL and non tumoral B cells, an analysis of their BCR repertoire has performed on FACS-sorting CD19+CD5-IgM-IgD- (non tumoral) and CD19+CD5+IgM-IgD- (tumoral) single B cells from blood samples of CLL patients with AIC. ZAP-70 positivity was screened by RT-PCR, and variable regions of heavy (IGVH) and light (IGVK/VL) immunoglobulin genes amplified by RT-PCR on ZAP-70+ cells. To date, analysis of 24 BCR sequences from 7 patients showed that non tumoral ZAP-70+ B cells were polyclonal, without stereotypy, using different V(D)J and CDR3 in comparison with those of the corresponding CLL B cells. IGVH of non tumoral ZAP-70+ B were mostly mutated, of replacement type, suggesting antigenic contact, contrary to CLL B cells. To determine potential autoreactivity of the non-tumoral ZAP-70+ B cells, IGVH and corresponding IGVK/VL were amplified for production of recombinant antibodies (rAb). To date, among 17 rAB from 7 different patients, 2/13 (15.4%) have an antinuclear autoreactivity on HEp-2 cells and 4/17 (23.5%) were polyreactive on ELISA (DNA, lipopolysaccharide, insulin), compared respectively to 6% and 4,3% of control B cells (Wardemann et al., Science 2003). Production of 7 additional rAb and tests for anti-erythrocytes and anti-platelets reactivity are in process. To study functional consequences of early ZAP-70 expression in B cells in vivo, we generated a knock in Zap-70+/Mb1-Cre+mouse model (KI ZAP), to induce conditional expression of ZAP-70 in the B cell compartment from the proB stage, with KI Zap-70+/Mb1-Cre-mice as controls (CTRL). The ZAP-70 mRNAs levels in B cells from KI ZAP mice were on average 20 times higher than that in CTRL B cells. Up to 20 months-old, KI ZAP mice did not develop signs of lymphoproliferation. KI ZAP mice had hypo-IgG since 16 weeks-old (p<0.001) together with hypo-IgM from 14 months-old (p<0.01). Immunophenotyping revealed a reduction in mature naive, mature switched as well as in germinal center B cells (p<0.001, p=0.002 and p<0.01 respectively) and a trend for plasma cells (p=0.07). Microarrays showed enrichment in circulating IgG and IgM autoantibodies against various antigens in KI ZAP mice. These mice had reduced apoptosis rates of proB (p<0.01), preB (p=0.02), and immatures B cells (p=0.03), together with enrichment in marginal zone (p=0.01), trend for transitional T2/T3, and reduction in B1a cells (p<0.01). After immunization by ovalbumin + Freund's adjuvant, a reduced production of specific IgG and IgM was observed (p=0.01 and p=0.03 respectively) with a trend in decreased number of antibody-secreting cells (p=0.07). KI ZAP B cells shown increased spontaneous activation and proliferation levels holding after BCR stimulation (p<0.01), as well as an increased intracellular calcic flow (p<0.001). Preliminary data suggested a reduced SYK phosphorylation after BCR stimulation in KI ZAP B cells. Our findings highlight for the first time that non tumoral B cells ZAP-70+ are distinct from CLL cells at cellular level, but probably enriched in autoreactive cells. Moreover, we shown that early ZAP-70 expression in normal B cells in vivois associated with autoimmune characteristics, together with partial block in B cells peripheral maturation, and a conversely early increased activation and proliferation status. ZAP-70 could interfere early with SYK leading to an altered BCR signaling responsible for defect in normal B maturation promoting emergence of autoreactive B cells. Mechanistic role of ZAP-70 in BCR signaling has to be further analyzed but our data open new opportunities involving ZAP-70 in the understanding of B cell development and physiopathology of tolerance breakdown. Disclosures No relevant conflicts of interest to declare.

scholarly journals Co-Expression of SYK and ZAP70 Subverts Negative B-Cell Selection and Enables Oncogenic Signaling in Multiple B-Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 295-295
Author(s):  
Teresa Sadras ◽  
Mickaël Martin ◽  
Lauren Kim-Sing ◽  
Jevon Cutler ◽  
Gal Lenz ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Cell Selection ◽  
B Cell Malignancies ◽  
Close Proximity ◽  
Mantle Cell ◽  
Bcr Signaling

B-cells are under intense selective pressure to eliminate autoreactive or premalignant clones. B-cell receptor (BCR) signals are required for survival, however, BCR-signaling exceeding maximum thresholds often reflects signaling from an autoreactive BCR or a transforming oncogene and triggers negative selection and cell death. The tyrosine kinase SYK initiates BCR-downstream signaling in B-cells while its close relative ZAP70 is almost exclusively expressed in T-cells. Interestingly, the segregation of SYK to B-cells and ZAP70 to T-cells is less confined in malignant lymphopoiesis suggesting that the balance of these related kinases may alter signaling output in disease and contribute to development of leukemia. As previously shown in B-cell chronic lymphocytic leukemia (B-CLL), we identified aberrant ZAP70 expression as a frequent feature in multiple other B-cell malignancies that depend on survival signals from a functional (pre-) BCR (E2A-PBX1+ pre-B ALL, and mantle cell lymphoma) or harbor oncogenic mimics of the BCR (BCR-ABL1+ B-ALL). Studying SYK and ZAP70 expression by single-cell Western blot, co-expression of the two tyrosine kinases was extremely rare in normal B- and T-cell populations. In contrast, &gt;50% of tumor B-cells in mantle cell lymphoma, pre-B ALL and CLL co-expressed SYK and ZAP70. Despite their structural similarities, genetic deletion and engineered reconstitution of SYK and ZAP70 in human B-cell lymphoma cells revealed striking functional differences. Proximity-dependent biotin identification (BioID) analyses identified that SYK, but not ZAP70, engaged the PI3K pathway via interaction with CD19. Consistent with this, reconstitution with SYK and SYK-ZAP70 but not ZAP70 alone promoted survival and proliferation. Detailed analysis of BCR-mediated cascades in lymphoma cells expressing SYK, ZAP70 or SYK-ZAP70 established that ZAP70 is only weakly efficient at propagating BCR-mediated calcium and downstream pathway activation in B-cells. Strikingly, co-expression of ZAP70 with SYK resulted in re-wired BCR-signaling of intermediate strength: compared to cells expressing only SYK, SYK-ZAP70 co-expressing cells had markedly reduced activation of the BLNK-BTK-PLCγ pathway, further reflected in BCR-induced Ca2+ signaling with delayed onset, lower amplitude but longer duration. In this way, we speculated that SYK and ZAP70 may be present within close proximity at the apex of BCR-initiated interactions, and hence compete for downstream substrates resulting in a re-wiring of classic signaling programs propagated normally by SYK. To explore this, we utilized proximity ligation assays (PLA) to monitor the proximity of SYK and ZAP70 in resting or BCR-stimulated B-cells, and found that SYK and ZAP70 co-exist within close proximity consistent with the view that varying levels of these kinases may alter B-cell signaling output. Functional experiments further showed that phosphomimetic activation of SYK, but not ZAP70, induced hyperactivation of PI3K-signaling and acute BTK-mediated cell death in pre-B ALL cells. In line with altered BCR-signaling strength and quality in SYK and ZAP70 co-expressing cells, over-expression of Zap70 in pre-B ALL cells rescued auto-immune checkpoint activation induced by hyper-activation of BCR-associated signaling. To study functional consequences of SYK-ZAP70 co-expression during normal B-cell development, we generated a novel knock in Zap-70+/Mb1-Cre+mouse model, to induce conditional expression of Zap70 in the B cell compartment from the proB stage. Consistent with compromised central tolerance checkpoints, Syk-Zap70 co-expressing pro/pre-B and immature B-cells had reduced spontaneous apoptosis rates and gave rise to autoantibody production against multiple self-antigens. Importantly, our findings highlight a previously unrecognized role for ZAP70 in oncogenic BCR-signaling and we conclude that the co-expression of ZAP70 mitigates the ability of SYK, downstream of an autoreactive BCR or a transforming oncogene, to trigger negative B-cell selection and cell death (Figure 1). Disclosures Weinstock: Celgene: Research Funding. Meffre:AbbVie: Consultancy, Other: Grant.

scholarly journals VLA-4 Expression and Activation in B Cell Malignancies: Functional and Clinical Aspects

2020 ◽  
Vol 21 (6) ◽  
pp. 2206 ◽  
Author(s):  
Andrea Härzschel ◽  
Antonella Zucchetto ◽  
Valter Gattei ◽  
Tanja Nicole Hartmann
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Types ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Clinical Aspects ◽  
Neoplastic Disease ◽  
B Cell Differentiation ◽  
B Cell Malignancies ◽  
Bcr Signaling

Lineage commitment and differentiation of hematopoietic cells takes place in well-defined microenvironmental surroundings. Communication with other cell types is a vital prerequisite for the normal functions of the immune system, while disturbances in this communication support the development and progression of neoplastic disease. Integrins such as the integrin very late antigen-4 (VLA-4; CD49d/CD29) control the localization of healthy as well as malignant B cells within the tissue, and thus determine the patterns of organ infiltration. Malignant B cells retain some key characteristics of their normal counterparts, with B cell receptor (BCR) signaling and integrin-mediated adhesion being essential mediators of tumor cell homing, survival and proliferation. It is thus not surprising that targeting the BCR pathway using small molecule inhibitors has proved highly effective in the treatment of B cell malignancies. Attenuation of BCR-dependent lymphoma–microenvironment interactions was, in this regard, described as a main mechanism critically contributing to the efficacy of these agents. Here, we review the contribution of VLA-4 to normal B cell differentiation on the one hand, and to the pathophysiology of B cell malignancies on the other hand. We describe its impact as a prognostic marker, its interplay with BCR signaling and its predictive role for novel BCR-targeting therapies, in chronic lymphocytic leukemia and beyond.

scholarly journals Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor-infiltrating nonmalignant B cells

Blood ◽  
2006 ◽  
Vol 108 (9) ◽  
pp. 3135-3142 ◽  
Author(s):  
Jonathan M. Irish ◽  
Debra K. Czerwinski ◽  
Garry P. Nolan ◽  
Ronald Levy
Keyword(s):  
B Cells ◽  
Cell Signaling ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cells ◽  
B Lymphoma Cells ◽  
Bcr Signaling ◽  
B Cell Subsets

Abstract The B-cell receptor (BCR) transmits life and death signals throughout B-cell development, and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20, Bcl-2, and BCR light chain isotype (κ or λ) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk, Syk, Erk1/2, and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells, achieved greater levels of per-cell signaling, and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention.

scholarly journals Autoimmunity Checkpoints As Therapeutic Targets in B-Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1587-1587
Author(s):  
Zhengshan Chen ◽  
Markus Muschen
Keyword(s):  
Drug Resistance ◽  
B Cells ◽  
B Cell ◽  
Negative Selection ◽  
Cell Types ◽  
Oncogenic Signaling ◽  
B Cell Malignancies ◽  
Minimum Threshold ◽  
Bcr Signaling ◽  
Novel Strategy

Abstract Concept. Targeted therapy of cancer typically focuses on inhibitors (e.g. tyrosine kinase inhibitors) that suppress oncogenic signaling below a minimum threshold required for survival and proliferation of cancer cells. Acute lymphoblastic leukemia (ALL) and B cell lymphomas originate from various stages of development of B cells, which unlike other cell types are under intense selective pressure. The vast majority of newly generated B cells are autoreactive and die by negative selection at autoimmunity checkpoints (AIC). Owing to ubiquitous encounter of self-antigen, autoreactive B cells are eliminated by overwhelming signaling strength of their autoreactive B cell antigen receptor (BCR). A series of recent findings suggests that, despite malignant transformation, AIC are fully functional in B cell malignancies. We propose that targeted engagement of AIC represents a previously unrecognized therapeutic opportunity to overcome conventional mechanisms of drug-resistance in pre-B ALL and other B cell malignancies. Results: Oncogenic drivers in B- cell malignancies function as mimics of B-cell receptor (BCR) signaling. Oncogenic activation of BCR-signaling represents the functional equivalent of positive selection during normal lymphocyte development. Addiction to survival and proliferation signals (or the equivalent of positive selection) is a common feature in many types of cancer. However, B-cell malignancies are unique in that they are also subject to an active negative selection process. B-cells expressing autoreactive BCRs or antibodies can cause systemic autoimmunity. As a safeguard against autoimmune diseases, lymphocyte development evolved autoimmunity checkpoints (AIC) to eliminate autoreactive clones. Owing to negative selection of autoreactive B-cells through AIC activation, lymphoid cells fundamentally differ in their signaling requirements from other cell types. Recent studies from our group showed that despite malignant transformation, B-cell leukemia and lymphoma cells are fully sensitive to negative selection and AIC-activation resulting (Chen et al., Nature 2015; Shojaee et al., Nature Med 2016; Chan et al., Nature 2017; Xiao et al., Cell 2018). AIC-activation in various lymphoid malignancies is achievable by pharmacological hyperactivation of BCR-signaling above a maximum threshold (see Schematic below). Unlike other types of cancer, B-cell malignancies are uniquely susceptible to clonal deletion induced by hyperactive signaling from an autoreactive BCR. Hence, targeted AIC-activation can be leveraged for eradication of drug-resistant leukemia and lymphoma clones. Here, we propose a novel strategy to overcome drug-resistance in B-lymphoid malignancies based on targeted activation of autoimmunity checkpoints (AIC) for removal of autoreactive B-lymphocytes. We have recently discovered that targeted hyperactivation of SYK, PI3K and ERK in B cell malignancies represents the functional equivalent of an autoimmunity checkpoint (AIC) for elimination of autoreactive clones among normal B cells. B cell tumors are uniquely vulnerable to AIC activation, suggesting that targeted activation of this checkpoint represents a novel strategy to induce cell death in otherwise drug-resistant B cell malignancies. Conclusion: Normal B-cells are positively selected for BCR signaling of intermediate strength (moderate activation of SYK, PI3K and ERK). In the absence of a functional BCR, SYK, PI3K and ERK activity fall below a minimum threshold, resulting in death by neglect. Hyperactivation above maximum thresholds (e.g. autoreactive BCR) triggers negative selection and cell death via AIC-activation. Targeted therapy of cancer typically focuses on agents that suppress oncogenic signaling below a minimum threshold. Our results support a novel strategy to overcome drug-resistance in B-cell malignancies based on targeted activation of autoimmunity checkpoints (AIC) for removal of autoreactive cells. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Development of a Transgenic Mouse Model to Study the Role of ZAP-70 in the Development and Progression of Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2830-2830
Author(s):  
Stefania Gobessi ◽  
Sara Bennardo ◽  
Pablo G Longo ◽  
Brendan Doe ◽  
Dimitar G Efremov
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mouse Model ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
Leukemia Development ◽  
Low Levels ◽  
The Impact

Abstract Abstract 2830 The protein tyrosine kinase ZAP-70 is an important prognostic factor in chronic lymphocytic leukemia (CLL). Patients that are considered ZAP-70-positive typically express 30–100% of the levels of ZAP-70 in T-cells, whereas in the remaining patients ZAP-70 is either not expressed or is expressed at lower levels. ZAP-70-positive patients have more aggressive disease and shorter survival than patients with low or absent ZAP-70. In vitro experiments with human lymphoma cell lines and primary CLL B-cells have shown that ZAP-70 is involved in B cell receptor (BCR) signaling, indicating that overexpression of ZAP-70 could affect the capacity of the leukemic cells to respond to antigen stimulation. Despite the strong association between ZAP-70 expression and prognosis, it is still not clear whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of more aggressive CLL. To further address this issue, we generated transgenic (tg) mice that express different levels of ZAP-70 in B cells. In these mice expression of the murine ZAP-70 transgene is targeted to the B cell compartment by a VH or a CD19 promoter (VH-ZAP70 and CD19-ZAP70 tg mice, respectively). B cells in CD19-ZAP70 tg mice express the same levels of ZAP-70 as normal murine T cells, whereas the levels of ZAP-70 in B cells of VH-ZAP70 tg mice are approximately 10 times lower. Immunophenotyping analysis of spleen and peritoneal cavity samples from wild type, VH-ZAP70 and CD19-ZAP70 tg mice did not reveal significant differences in the percentage of follicular (FO), marginal zone (MZ) and B1 B cells, indicating that ectopic expression of ZAP-70 does not affect normal B cell development and maturation. In terms of BCR signal transduction, no abnormalities were detected in VH-ZAP70 tg mice, suggesting that low levels of ZAP-70 do not affect BCR signaling. In contrast, B cells from CD19-ZAP70 tg mice showed altered phosphorylation of several molecules downstream of the BCR, such as Syk and BLNK, whereas phosphorylation of Cbl was not affected. To investigate the impact of ZAP-70 expression on leukemia development and progression, we crossed VH-ZAP70 and CD19-ZAP70 tg mice with Eμ-TCL1 tg mice. The latter mice develop leukemias that are considered a mouse model of human CLL. These leukemias are CD5+, express unmutated IGHV genes and stereotyped polyreactive BCRs, but are always ZAP-70-negative. VH-ZAP70/Eμ-TCL1 tg mice (n=11) have been followed for over a year and did not show any differences with respect to their Eμ-TCL1 littermates (n=10). Both groups, starting from the age of 7–8 months, developed leukemias with a similar rate of progression and impact on survival, suggesting that low levels of ZAP-70 do not affect the behavior of the disease. The cohort of CD19-ZAP70/Eμ-TCL1 tg mice was more recently established. These animals are currently 4 months old and still do not show signs of leukemia development. Data from the extended follow-up of these mice will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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